Without limiting the scope of the invention, its background is described in connection with the use of RT enzymes for use in a wide variety of assays used by molecular biologists, as an example.
Heretofore, in this field, the RNA directed, DNA polymerase activity responsible for the synthesis of complementary DNA (cDNA) from an RNA template has been accomplished using reverse transcriptase enzymes, whether purified or recombinant. One such use for RTs if for transcription-based amplification systems, e.g., amplifying RNA and DNA target sequences.
Transcription-based amplification methods find use in a wide variety of settings, e.g., routine clinical laboratory use in diagnostic tests such as direct detection of pathogens. Another such use for RTs is in the initial step for RT-PCR (polymerase chain reaction) used to amplify an RNA target for analysis and/or cloning. In RT-PCR, the RT is used to make an initial complementary DNA (cDNA) copy of the RNA target, which is then amplified by successive rounds of DNA replication.
RTs have three primary enzymatic activities: a RNA-directed, DNA polymerase activity; a DNA-directed, DNA polymerase activity; and an RNase H activity. The RNase H activity degrades specifically RNA found in an RNA:DNA duplex. An initial goal of many molecular biologists was to identify an RT that had no detectable RNase H activity while still maintaining DNA polymerase activity. An RT having no RNase H activity would finds particular use because degradation of the RNA strand of RNA:DNA intermediates by RNase H causes unwanted degradation of the template reducing cDNA yields. U.S. Pat. Nos. 5,244,797, 5,540,776, 5,668,005, 6,063,608, 6,589,768 and 6,610,522, disclose one such mutant RT, wherein a gross deletion mutant with no detectable RNase H activity is taught.
U.S. Pat. No. 5,998,195 teaches a method of reducing the level of RNase activity in an RT preparation by using an expression vector or plasmid containing a cloned version of the gene for an MMLV-RT which, when used to transform a suitable host cell such as E. coli, leads to the expression of the gene and the generation of a gene product having the DNA- and RNA-directed DNA polymerase activities and RNase H activity associated with retroviral reverse transcriptases. A host cell with a reduced level of ribonuclease activity as compared to wild-type strains is used to provide a source of RT that has endogenous levels of RNase activity below that of previous recombinant preparations.
Yet others have purified RT, e.g., Goff et al., U.S. Pat. No. 4,943,531 (1990) and Kotewicz et al., U.S. Pat. No. 5,017,492, which have described methods for the purification of reverse transcriptase derived from Moloney Murine Leukemia Virus (MMLV-RT) and expressed in E. coli. These expression constructs and isolation and purification methods form the basis for the majority of commercial reverse transcriptase preparations.